This invention relates to a method for the immobilization of enzymes onto siliceous support materials and the use of such immobilized enzymes in continuous enzyme-based processes such as the production of pharmaceuticals. Immobilized enzymes prepared according to the invention have potential applications, however, in a wide range of synthetic and materials treatment processes such as the production of specialty commodity chemicals, waste water treatment and pulp and paper processing.
The industrial use of enzymes is often limited by their high cost and rapid inactivation. In particular, the use of soluble enzymes necessitates regular replenishment of the enzymes, lost with the product at the conclusion of a process. To improve their economic feasibility in industrial processes, enzymes are generally immobilized onto a matrix. Immobilization facilitates re-use of the enzymes, and may affect the selectivity and stability of the enzyme. Immobilization research has focused upon means to enhance the transfer of enzymes onto the support, and upon means to ensure that the transferred enzymes remain active.
A number of different organic and inorganic support matrices and enzyme immobilization techniques have been tried with a view to achieving a high level of enzyme uptake with a minimum of enzyme degradation or inactivation. One such approach is the immobilization of an enzyme by its physical entrapment within a gel, microcapsule or similar polymeric structure. An example is afforded by U.S. Pat. No. 3,850,751 (Messing) which teaches the adsorption of an enzyme to the inner surface of a porous, essentially non-siliceous ceramic body having an average pore diameter at least as large as the largest dimension of the enzyme.
While entrapment is a simple process and generally affords a high uptake of enzyme without appreciable inactivation during the immobilization process, the enzyme once bound is surrounded by a matrix imposing a mass transfer barrier. In the result, the observed activity may be much lower than the intrinsic activity of the enzyme. On the other hand, direct physical adsorption of the enzyme to a substrate, without any entrapment, is generally characterized by relatively weak binding between enzyme and support, leading to significant enzyme desorption.
Another approach is the direct covalent bonding of an enzyme to a suitably chemically modified support medium. While this leads to strong bonding between the enzyme and the support, a labour-intensive and expensive multi-step procedure is usually involved (including the step of "activating" the support). Too, low enzyme yields are not uncommon, owing to inactivation of the enzyme by the harsh conditions employed in the immobilization process.
A further, widely used approach to enzyme immobilization might be referred to as the "covalent cross-linking" process and is exemplified by U.S. Pat. Nos. 4,071,409 (Messing et al.); 4,258,133 (Mirabel et al.); and 4,888,285 (Nishimura et al.). According to the teachings of these patents a support medium is modified or coated to present functionalities which can then be linked by way of a cross-linking agent to free functional groups on the enzyme. Thus, Nishimura et al. modifies a silica gel or porous glass support surface by reaction with an aminosilane derivative in an organic solvent. The resulting aminated support is then linked to the enzyme in the presence of a polyfunctional cross-linking agent (glutaraldehyde), a phenoxycarboxylic acid (tannic acid) and, optionally, a basic polysaccharide (e.g. chitosan). Nishimura et al. asserts that the tannic acid and chitosan stabilize the enzyme, so as to reduce inactivation by the cross-linking agent during the immobilization process.
According to the aforementioned Mirabel patent, which affords a second example of the covalent cross-linking technique, an inorganic support having surface hydroxyl groups (e.g. brick, alumina, aluminosilicates) is modified with compounds containing an alcohol or phenol group (e.g. monoethanolamine, amino-1 pentanol, p-aminophenol) to generate an ester linkage on a "grafted" support. The resulting grafted support is then coupled to the enzyme, usually in the presence of a bifunctional reagent.
Known enzyme immobilization proceedings employing covalent cross-linking involve in many cases, time consuming modifications to the substrate surface and/or the use of expensive or hazardous reagents (either solvents or the grafting agents themselves).
It is an object of the present invention to provide a simple and efficient method for the immobilization of enzymes on siliceous supports, requiring no prior modification of the support material to avoid the disadvantages attendant on such modification. Our approach is based on the discovery that polyaldehyde cross-linking agents may be used to immobilize enzymes onto previously unmodified siliceous support materials, to produce water-insoluble immobilized enzyme complexes exhibiting high yields of enzyme activity and stability.
It is a further object of the invention to provide immobilized enzymes which may usefully be applied to continuous enzymatic reactions with a variety of industrial applications, including waste water treatment, production of pharmaceuticals and other commodity chemicals, and pulp and paper processing.